Program Description/Abstract A central component of peripheral immune tolerance is Foxp3[+] regulatory T cells (Tregs). Tregs are indispensable for the prevention of autoimmune diseases, but also serve as a major hurdle to tumor immunity and immunotherapy. IL-2 is considered a major regulator for controlling the homeostasis and more recently, lineage stability, of Tregs by signaling through STAT5. Recent studies have also discovered a highly suppressive p-STAT5[+] Treg subpopulation that is critical for the suppression of autoreactive T cells and incipient autoimmunity. Of note, low-dose IL-2 specifically activates Tregs to ameliorate autoimmune diseases in murine models and clinical trials, and there is a growing interest in exploring this new therapeutic strategy. Unlike conventional T cells, Tregs are normally kept in a state of partial IL-2 deficiency due to, in part, Foxp3-dependent repression of IL-2 production and are therefore indexed to a low IL-2 signaling threshold. Additionally, Tregs show a predominant requirement of STAT5 activity due to their low PI3K and MAPK activities downstream of IL-2R. Mechanisms underlying Treg-specific regulation of IL-2 and STAT5 signaling remain uncertain. Through a kinase inhibitor screen, we identified Mst1, a core kinase in Hippo signaling, as a novel IL-2 signal sensor to amplify STAT5 activation in Tregs but not conventional T cells. We therefore hypothesize that Hippo kinases selectively sense and amplify IL-2R?STAT5 signaling to adapt Tregs to a proper IL-2 signaling threshold, thereby maintaining a stable Treg population and regulatory activity. Aim 1. Establish Mst1?STAT5 axis in Tregs under homeostasis and activation in vivo. Aim 2. How does Hippo/Mst1 signal in Tregs? Aim 3. Define and reconstruct IL-2-dependent signaling circuits in Tregs. We predict our studies will establish a new paradigm in Treg biology and immune regulation, as well as new mechanisms of Hippo signaling, with the potential to translate into innovative strategies to target autoimmunity and cancer.